Effect of Feed Restriction on Performance and Postprandial Nutrient Metabolism in Pigs Co-Infected with Mycoplasma hyopneumoniae and Swine Influenza Virus

As nutritional status and inflammation are strongly connected, feeding and nutritional strategies could be effective to improve the ability of pigs to cope with disease. The aims of this study were to investigate the impact of a feed restriction on the ability of pigs to resist and be tolerant to a coinfection with Mycoplasma hyopneumoniae (Mhp) and the European H1N1 swine influenza virus, and the consequences for nutrient metabolism, with a focus on amino acids. Two groups of specific pathogen-free pigs were inoculated with Mhp and H1N1 21 days apart. One group was fed ad libitum, the other group was subjected to a two-week 40% feed restriction starting one week before H1N1 infection. The two respective mock control groups were included. Three days post-H1N1 infection, 200 g of feed was given to pigs previously fasted overnight and serial blood samples were taken over 4 hours to measure plasma nutrient concentrations. Throughout the study, clinical signs were observed and pathogens were detected in nasal swabs and lung tissues. Feed-restricted pigs presented shorter hyperthermia and a positive mean weight gain over the 3 days post-H1N1 infection whereas animals fed ad libitum lost weight. Both infection and feed restriction reduced postprandial glucose concentrations, indicating changes in glucose metabolism. Post-prandial plasma concentrations of the essential amino acids histidine, arginine and threonine were lower in co-infected pigs suggesting a greater use of those amino acids for metabolic purposes associated with the immune response. Altogether, these results indicate that modifying feeding practices could help to prepare animals to overcome an influenza infection. Connections with metabolism changes are discussed.     

Asymmetric distribution of phospholipids in spectrin-poor erythrocyte vesicles

We have investigated by electron spin resonance, at 37 degrees C, the outside-inside passage and the equilibrium distribution of spin-labeled phospholipids, respectively, in ATP-containing ghosts, in heat-treated erythrocytes, and in heat-induced vesicles. The heat-treated vesicles were spectrin depleted to approximately 25% of the original content and had lost almost 100% of the other cytoskeletal proteins. Yet the vesicles, as long as they contained ATP, were capable of translocating the aminophospholipids with the same efficiency as the heat-treated erythrocytes, and almost with the same efficiency as ATP-containing ghosts. In the vesicles, sphingomyelin and phosphatidylcholine analogues underwent a very slow transverse diffusion as in native cells. We conclude that spectrin and other cytoskeleton proteins are not major factors for the establishment and maintenance of phospholipid asymmetry in human erythrocytes, which may be chiefly due to the aminophospholipid translocase activity.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

Progress of developmental programme during the last larval instar of Bombyx mori: Relationships with food intake,ecdysteroids and juvenile hormone

The progress of developmental programme in the epidermal cells of last instar larvae of Bombyx mori was determined by ecdysteroid injections in normal and in JH-treated larvae. To clarify the importance of food intake in the control of development, starved animals were also used.The instar begins with a period during which the larval programme is expressed: this occurs in the presence of 20-hydroxyecdysone. Epidermal cells can thereafter secrete pupal cuticle after ecdysteroid injection although the larval programme is normally still present. During the last period only pupal characters can be expressed either in normal or in 20-hydroxyecdysone-injected larvae.These different developmental phases are not correlated with obligatory and facultative feeding periods.Transition from the first to the second phases is correlated with the absence of JH effects on pupal genes. JH applications during the second period, however, prevent the expression of pupal characters after 20-hydroxyecdysone injection. Thus, during this period, the pupal programme is not stabilized. Cellular reprogramming itself occurs at the onset of the last developmental period and is probably under the control of ecdysteroids.     

In Situ Fe and S isotope analyses in pyrite from the 3.2 Ga Mendon Formation (Barberton Greenstone Belt,South Africa): Evidence for early microbial iron reduction

On the basis of phylogenetic studies and laboratory cultures, it has been proposed that the ability of microbes to metabolize iron has emerged prior to the Archaea/Bacteria split. However, no unambiguous geochemical data supporting this claim have been put forward in rocks older than 2.7–2.5 giga years (Gyr). In the present work, we report in situ Fe and S isotope composition of pyrite from 3.28‐ to 3.26‐Gyr‐old cherts from the upper Mendon Formation, South Africa. We identified three populations of microscopic pyrites showing a wide range of Fe isotope compositions, which cluster around two δ⁵⁶Fe values of ?1.8‰ and +1‰. These three pyrite groups can also be distinguished based on the pyrite crystallinity and the S isotope mass‐independent signatures. One pyrite group displays poorly crystallized pyrite minerals with positive Δ³³S values > +3‰, while the other groups display more variable and closer to 0‰ Δ³³S values with recrystallized pyrite rims. It is worth to note that all the pyrite groups display positive Δ³³S values in the pyrite core and similar trace element compositions. We therefore suggest that two of the pyrite groups have experienced late fluid circulations that have led to partial recrystallization and dilution of S isotope mass‐independent signature but not modification of the Fe isotope record. Considering the mineralogy and geochemistry of the pyrites and associated organic material, we conclude that this iron isotope systematic derives from microbial respiration of iron oxides during early diagenesis. Our data extend the geological record of dissimilatory iron reduction (DIR) back more than 560 million years (Myr) and confirm that micro‐organisms closely related to the last common ancestor had the ability to reduce Fe(III).     

Divergent selection on flowering phenology but not on floral morphology between two closely related orchids

Closely related species often differ in traits that influence reproductive success, suggesting that divergent selection on such traits contribute to the maintenance of species boundaries. Gymnadenia conopsea ss. and Gymnadenia densiflora are two closely related, perennial orchid species that differ in (a) floral traits important for pollination, including flowering phenology, floral display, and spur length, and (b) dominant pollinators. If plant–pollinator interactions contribute to the maintenance of trait differences between these two taxa, we expect current divergent selection on flowering phenology and floral morphology between the two species. We quantified phenotypic selection via female fitness in one year on flowering start, three floral display traits (plant height, number of flowers, and corolla size) and spur length, in six populations of G. conopsea s.s. and in four populations of G. densiflora. There was indication of divergent selection on flowering start in the expected direction, with selection for earlier flowering in two populations of the early‐flowering G. conopsea s.s. and for later flowering in one population of the late‐flowering G. densiflora. No divergent selection on floral morphology was detected, and there was no significant stabilizing selection on any trait in the two species. The results suggest ongoing adaptive differentiation of flowering phenology, strengthening this premating reproductive barrier between the two species. Synthesis: This study is among the first to test whether divergent selection on floral traits contribute to the maintenance of species differences between closely related plants. Phenological isolation confers a substantial potential for reproductive isolation, and divergent selection on flowering time can thus greatly influence reproductive isolation and adaptive differentiation.     

Reduction in the metabolic levels due to phenotypic plasticity in the Pyrenean newt,Calotriton asper,during cave colonization

According to theories on cave adaptation, cave organisms are expected to develop a lower metabolic rate compared to surface organisms as an adaptation to food scarcity in the subterranean environments. To test this hypothesis, we compared the oxygen consumption rates of the surface and subterranean populations of a surface‐dwelling species, the newt Calotriton asper, occasionally found in caves. In this study, we designed a new experimental setup in which animals with free movement were monitored for several days in a respirometer. First, we measured the metabolic rates of individuals from the surface and subterranean populations, both maintained for eight years in captivity in a natural cave. We then tested individuals from these populations immediately after they were caught and one year later while being maintained in the cave. We found that the surface individuals that acclimated to the cave significantly reduced their oxygen consumption, whereas individuals from the subterranean population maintained in the cave under a light/dark cycle did not significantly modify their metabolic rates. Second, we compared these metabolic rates to those of an obligate subterranean salamander (Proteus anguinus), a surface aquatic Urodel (Ambystoma mexicanum), and a fish species (Gobio occitaniae) as references for surface organisms from different phyla. As predicted, we found differences between the subterranean and surface species, and the metabolic rates of surface and subterranean C. asper populations were between those of the obligate subterranean and surface species. These results suggest that the plasticity of the metabolism observed in surface C. asper was neither directly due to food availability in our experiments nor the light/dark conditions, but due to static temperatures. Moreover, we suggest that this adjustment of the metabolic level at a temperature close to the thermal optimum may further allow individual species to cope with the food limitations of the subterranean environment.     

Possible Links Between Stress Defense and the Tricarboxylic Acid (TCA) Cycle in Francisella Pathogenesis

Francisella tularensis is a highly infectious bacterium causing the zoonotic disease tularemia. In vivo, this facultative intracellular bacterium survives and replicates mainly in the cytoplasm of infected cells. We have recently identified a genetic locus, designated moxR that is important for stress resistance and intramacrophage survival of F. tularensis. In the present work, we used tandem affinity purification coupled to mass spectrometry to identify in vivo interacting partners of three proteins encoded by this locus: the MoxR-like ATPase (FTL_0200), and two proteins containing motifs predicted to be involved in protein–protein interactions, bearing von Willebrand A (FTL_0201) and tetratricopeptide (FTL_0205) motifs. The three proteins were designated here for simplification, MoxR, VWA1, and TPR1, respectively. MoxR interacted with 31 proteins, including various enzymes. VWA1 interacted with fewer proteins, but these included the E2 component of 2-oxoglutarate dehydrogenase and TPR1. The protein TPR1 interacted with one hundred proteins, including the E1 and E2 subunits of both oxoglutarate and pyruvate dehydrogenase enzyme complexes, and their common E3 subunit. Remarkably, chromosomal deletion of either moxR or tpr1 impaired pyruvate dehydrogenase and oxoglutarate dehydrogenase activities, supporting the hypothesis of a functional role for the interaction of MoxR and TPR1 with these complexes. Altogether, this work highlights possible links between stress resistance and metabolism in F. tularensis virulence.Francisella tularensis is responsible for the disease tularamia in a large number of animal species. This highly infectious bacterial pathogen can be transmitted to humans in numerous ways (1, 2, 3), including direct contact with sick animals, inhalation, ingestion of contaminated water or food, or by bites from ticks, mosquitoes, or flies. Four different subspecies (subsp.) of F. tularensis that differ in virulence and geographic distribution exist, designated subsp. tularensis (type A), subsp. holarctica (type B), subsp. Novicida, and subsp. mediasiatica, respectively. F. tularensis subsp. tularensis is the most virulent subspecies causing a severe disease in humans, whereas F. tularensis subsp. holarctica causes a similar disease but of less severity (4). Because of its high infectivity and lethality, F. tularensis is considered a potential bioterrorism agent (5).F. tularensis is able to survive and to replicate in the cytoplasm of a variety of infected cells, including macrophages. To resist this stressful environment, the bacterium must have developed stress resistance mechanisms, most of which are not yet well characterized. We recently reported the identification of a novel genetic locus that is important for stress resistance and intracellular survival of F. tularensis (6). This locus was designated moxR because the first gene FTL_0200, encodes a protein belonging to the AAA+ ATPase of the MoxR family ((7) and references therein). The data obtained in that first study had led us to suggest that the F. tularensis MoxR-like protein might constitute, in combination with other proteins of the locus, a chaperone complex contributing to F. tularensis pathogenesis.To further validate this hypothesis and expand our initial observations, we here decided to perform tandem affinity purification (TAP),¹ using a dual affinity tag approach coupled to mass spectroscopy analyses (8), to identify proteins interacting in vivo with three proteins encoded by the proximal portion of the moxR locus. For this, we chose as baits: the MoxR-like protein (FTL_0200) and two proteins bearing distinct motifs possibly involved in protein–protein interactions, FTL_0201 (Von Willebrand Factor Type A domain, or VWA) and FTL_0205 (tetratrichopeptide repeat or TPR). The three proteins were designated here for simplification, MoxR, VWA1, and TPR1; and the corresponding genes moxR, vwa1, and tpr1, respectively.VWA domains are present in all three kingdoms of life. They consist of a β-sheet sandwiched by multiple α helices. Frequently, VWA domain-containing proteins function in multiprotein complexes (9). TPR typically contain 34 amino acids. Many three-dimensional structures of TPR domains have been solved, revealing amphipathic helical structures (10). TPR-containing proteins are also found in all kingdoms of life. They can be involved in a variety of functions, and generally mediate protein–protein interactions. In the past few years, several TPR-related proteins have been shown to be involved in virulence mechanisms in pathogenic bacteria ((11) and references therein).Our proteomic approach allowed us to identify a series of protein interactants for each of the three moxR-encoded proteins. Remarkably, the protein TPR1 interacted with all the subunits of the pyruvate dehydrogenase (PDH) and 2-oxoglutarate dehydrogenase (OGDH) complexes. Furthermore, inactivation of tpr1 also severely impaired the activities of these two enzymes. Inactivation of tpr1 affected bacterial resistance to several stresses (and in particular oxidative stress), intramacrophagic bacterial multiplication and bacterial virulence in the mouse model. Functional implications and possible relationship between bacterial metabolism, stress defense, and bacterial virulence are discussed.     

Silibinin inhibits hepatitis C virus entry into hepatocytes by hindering clathrin‐dependent trafficking

Hepatitis C virus (HCV) is a global health concern infecting 170 million people worldwide. Previous studies indicate that the extract from milk thistle known as silymarin and its main component silibinin inhibit HCV infection. Here we investigated the mechanism of anti‐HCV action ofsilymarin‐derived compounds at the molecular level. By using live‐cell confocal imaging, single particle tracking, transmission electron microscopy and biochemical approaches on HCV‐infected human hepatoma cells and primary hepatocytes, we show that silibinin potently inhibits HCV infection and hinders HCV entry by slowing down trafficking through clathrin‐coated pits and vesicles. Detailed analyses revealed that silibinin altered the formation of both clathrin‐coated pits and vesicles in cells and caused abnormal uptake and trafficking of transferrin, a well‐known cargo of the clathrin endocytic pathway. Silibinin also inhibited infection by other viruses that enter cells by clathrin‐mediated endocytosis including reovirus, vesicular stomatitis and influenza viruses. Our study demonstrates that silibinin inhibits HCV early steps of infection by affecting endosomal trafficking of virions. It provides new insights into the molecular mechanisms of action of silibinin against HCV entry and also suggests that silibinin is a potential broad‐spectrum antiviral therapy.